Persistent cytosolic expression of Smac56 attenuates its interaction with Apollon and cIAPs.

<p>Cells indicated by a <i>T</i> (Transient) were cotransfected with Ub-Smac56-V5 (IRES2.eGFP), whereas those indicated by a <i>S</i> (Stable) stably expressed Ub-Smac56-V5 (IRES2.eGFP). Additional cultures were transiently transfected with the indicated FLAG-IAP (p3XFLAG.CMV10). The cells that stably expressed Ub-Smac56-V5 were treated for 12 h with 20 µM zIEALal to increase the level of Smac56, which is rapidly degraded by UPS (Burke and Smith, unpublished data). Cell pellets from each IAP transfection were combined with a pellet of cells that were transiently or stably transfected with Ub-Smac56. The combined cell pellets were lysed, and proteins were immunoprecipitated with a polyclonal antibody to the V5 epitope. Lysate proteins (<i>Input</i>) and proteins eluted from the immune complex (<i>Immunoprecipitate</i>) were resolved by SDS-PAGE and subjected to western blot analysis with monoclonal antibody to the V5 or FLAG epitope.